Gangliosides (sialylated glycosphingolipids) are ubiquitous components of mammalian cells. Ganglioside expression is altered in many tumor types, particularly in malignant melanoma, neuroblastoma, and astrocytoma. Gangliosides also serve as important antigens in these tumor types. While the pattern of expression of gangliosides in tumors is now well understood, very little is known about what determines the characteristic patterns in different cell types and the altered expression following transformation. The main goal of this project is to understand the ways that ganglioside expression in tumor cells is determined and regulated. The emphasis will be on an analysis of the various glycosyltransferases that are responsible for the biosynthesis of gangliosides in malignant melanoma and neuroblastoma cells. We have previously studied glycosyltransferase levels and activities in a panel of cells lines and identified key steps in the biosynthesis of gangliosides in these cells. GD3 synthase (SAT-2) and GM2\GD2 synthase (GalNAc-T) were particularly important enzymes in this process. We have recently isolated a cDNA clone coding for GalNAc-T and studied some of its properties. These studies will be extended by (i) continuing to characterize;, the GalNAc-T gene and its product, (ii) producing antibodies to this enzyme, (iii) using these antibodies to isolate the enzyme and to examine its glycosylation, (iv) attempt to clone a cDNA for SAT-2, (v) purify this enzyme and study its properties, and (vi) produce antibodies to SAT-2. Using these reagents we will be in a position to begin a more detailed analysis of the control mechanisms that determine the ganglioside makeup of these tumors. The factors involved will include transcriptional or translational control in determining enzyme levels and activity, the role of competition among the different glycosyltransferase for common substrates, the influence of glycosidases, and the sub-cellular organization of the enzymes. Ultimately this information could form the basis for altering the antigenicity or behavior of tumor cells.